Short-Chain Fatty-Acid-Producing Bacteria: Key Components of the Human Gut Microbiota

Short-chain fatty acids (SCFAs) play a key role in health and disease, as they regulate gut homeostasis and their deficiency is involved in the pathogenesis of several disorders, including inflammatory bowel diseases, colorectal cancer, and cardiometabolic disorders. SCFAs are metabolites of specific bacterial taxa of the human gut microbiota, and their production is influenced by specific foods or food supplements, mainly prebiotics, by the direct fostering of these taxa. This Review provides an overview of SCFAs’ roles and functions, and of SCFA-producing bacteria, from their microbiological characteristics and taxonomy to the biochemical process that lead to the release of SCFAs. Moreover, we will describe the potential therapeutic approaches to boost the levels of SCFAs in the human gut and treat different related diseases.


Introduction on Human Gut Microbiota
The human gut microbiota comprises an ecological community that includes bacteria, yeasts, viruses and parasites, yielding nearly 100 trillion microorganisms [1][2][3][4]. At birth, the human gut is almost sterile, and is soon populated by the mother microbiome. The type of delivery, vaginal or cesarean, plays a main role in the composition of the gut microbiome in the newborn [4]. Healthy gut microbiota mainly comprises (nearly 90%) two phyla, Firmicutes and Bacteroidetes, and also contain less-represented phyla, such as Proteobacteria, Verrucomicrobia, or Actinobacteria [5]. The phylum Firmicutes includes several genera, of which the most common (up to 95% of the total) are Lactobacillus, Bacillus, Enterococcus, Ruminicoccus and Clostridium [5]. the Bifidobacterium genus is the most abundant of the Actinobacteria phyla; however, the phylum itself is less present overall [5]. Many of the microorganisms in the normal microbiota are opportunistic pathogens [6,7]. The distinction between opportunistic pathogens and commensal microbes is largely determined by the host immunity, rather than any intrinsic feature of that microorganism. Therefore, competent human immunity can shape potential pathogenic microbes into symbiotic ones. Microbes also colonize other sites of the gastrointestinal tract, and the composition of the esophageal or gastric microbiome differs from that of the microbiome in the gut [8,9].
Intestinal microbiota is the main source of bacteria, producing SCFAs through the degradation of substrates, mainly non-digestible polysaccharides, including dietary fibers and resistant starches. Interestingly, the concentration of SCFA fluctuates throughout our life, and these longitudinal changes appear to be related to the composition of our gut microbiome, which also varies during our life cycle [64,65]. Notably, the variety of our diet, which changes during our life, has a heavy influence on the quantity of SCFAs released in the intestine, modulating the amount of substrate sources for SCFAs-producing bacteria [66].
Specifically, in early life, the microbiota evolves from being dominated by Enterobacteriaceae to being dominated by Bifidobacteriaceae while later, along with the end of breastfeeding, an increase in the abundance of Firmicutes can be observed [67,68]. Firmicutes species, including Lactobacillaceae, Ruminococcaceae and Lachnospiraceae, are able to break down complex polysaccharides and other sugars through hydrolysis, resulting in the production of butyrate and other SCFAs [69,70]. Finally, in older age, the microbiota changes again and the abundance of Enterobacteriaceae increases [68,71]. These changes in the microbiota are also reflected in the production and diversity of SCFA, or even in the profile of branched short-chain fatty acids (BCFA) [72,73], as shown in Figure 1.
the microbiota are also reflected in the production and diversity of SCFA, or even in the profile of branched short-chain fatty acids (BCFA) [72,73], as shown in Figure 1. The levels of the SCFAs detected in the gut, acetate, propionate and butyrate are affected by these age-driven changes in the human gut microbiota. Acetate concentration is higher in the early stages of life as it is the main product of Bifidobacteria strains that characterise the infant gut microbiota: strains B. bifidum, B. infantis and B. breve are the main players [68,74] Bifidobacterium strains are able to utilize the human milk oligosaccharides (HMOs) [75] to obtain carbon and energy [76]. HMOs have galactose and glucose as their main components and Bifidobacteriaceae can transform galactose into glucose. The consumption of this glucose is associated with an increase in acetate and formate, 1,2-propanediol, and lactate, as has been seen by the increase in such metabolites during in vitro Bifidobacterium co-cultivation [77].
Healthy infant microbiota development is characterized by the predominance of certain Bifidobacterium species, and the absence of HMO-metabolizing bifidobacterial genes has been correlated with systemic and intestinal inflammation [78,79]. In addition, in vitro studies suggest that acetate-producing Bifidobacterium is able to protect against bacterial infections, as has been observed with pathogenic E. coli [80]. Interestingly, the produced acetate benefits the growth of propionate and butyrate-producing bacteria and, at the same time, butyrate favors the growth of Bifidobacterium, leading to a cross-feeding between SCFA-producing bacteria [72,81].
Gut propionate levels increase after the cessation of breastfeeding and the introduction of a more varied diet. Due to the presence of more diverse food, the microbiota change, with a greater proportion of Firmicutes, mainly of the Clostridia class [73,84]. Propionate can be formed from the fermentation of sugars in three ways. The succinate pathway processes hexoses and pentoses (via a pathway that, thanks to vitamin B12, converts succinyl-Coa into proprionate), while the acrylate pathway converts lactate into propionate, and, through the propanediol pathway, deoxy sugars (e.g., fucose and The levels of the SCFAs detected in the gut, acetate, propionate and butyrate are affected by these age-driven changes in the human gut microbiota. Acetate concentration is higher in the early stages of life as it is the main product of Bifidobacteria strains that characterise the infant gut microbiota: strains B. bifidum, B. infantis and B. breve are the main players [68,74] Bifidobacterium strains are able to utilize the human milk oligosaccharides (HMOs) [75] to obtain carbon and energy [76]. HMOs have galactose and glucose as their main components and Bifidobacteriaceae can transform galactose into glucose. The consumption of this glucose is associated with an increase in acetate and formate, 1,2propanediol, and lactate, as has been seen by the increase in such metabolites during in vitro Bifidobacterium co-cultivation [77].
Healthy infant microbiota development is characterized by the predominance of certain Bifidobacterium species, and the absence of HMO-metabolizing bifidobacterial genes has been correlated with systemic and intestinal inflammation [78,79]. In addition, in vitro studies suggest that acetate-producing Bifidobacterium is able to protect against bacterial infections, as has been observed with pathogenic E. coli [80]. Interestingly, the produced acetate benefits the growth of propionate and butyrate-producing bacteria and, at the same time, butyrate favors the growth of Bifidobacterium, leading to a cross-feeding between SCFA-producing bacteria [72,81].
Acetate production requires substrates described as acetogenic fibers (inulin, galactoligosaccharides, etc.) [66]. Those fibers may then enter two possible pathways: acetogenesis or carbon fixation pathway. Acetogenesis is the production of acetate, mediated by homoacetogenic bacteria, which can use both H 2 and CO 2 , while the carbon-fixation pathway produces acetate directly from CO 2 [66,82,83].
Gut propionate levels increase after the cessation of breastfeeding and the introduction of a more varied diet. Due to the presence of more diverse food, the microbiota change, with a greater proportion of Firmicutes, mainly of the Clostridia class [73,84]. Propionate can be formed from the fermentation of sugars in three ways. The succinate pathway processes hexoses and pentoses (via a pathway that, thanks to vitamin B12, converts succinyl-Coa into proprionate), while the acrylate pathway converts lactate into propionate, and, through the propanediol pathway, deoxy sugars (e.g., fucose and rhamnose) are processed [85]. Bacteria that use the succinate route belong mostly to the Bacteroidetes (Prevotella spp.) and Negativicutes classes, such as Phascolarctobacterium succinatutens or Veillonella spp. [86][87][88]. In the case of the acrylate route, the best-studied bacteria are Coprococcus spp. which belongs to the Lachnospiraceae family. Strikingly, some of the members of this genus are capable of producing butyrate in addition to propionate, depending on the initial substrate used [85]. Finally, propanediol-dependent metabolic pathways have been observed in Roseburia inilivorans and in Blautia species, which also belong to the Lachnospiraceae family [89,90].
Among the three main SCFAs, butyrate has the greatest impact at the physiological level; therefore, the bacteria that produce this have the highest relevance.
Butyrate derives from the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA, which is then gradually reduced to butyryl-CoA. Butyryl-CoA is then transformed in butyrate via butyryl-CoA:acetate CoA-transferase or via phosphotransbutyrylase and butyrate kinase [89].
Butyrate-producing microbial communities in the gut are essential for maintaining a healthy gut environment. These communities play a critical role in limiting the entry and establishment of other bacteria, particularly harmful pathogens. The production of butyrate by these bacteria is necessary for the colonocytes to generate energy and to increase epithelial oxygen consumption [91,92]. This, in turn, helps to maintain an anaerobic gut environment that is harsh for opportunistic aerobic pathogens such as Salmonella and E. coli [93,94]. As previously mentioned, the main species involved in the production of butyrate are found within the Lachnospiraceae and Ruminococcaceae families [95,96]. As an example of the Lachnospiraceae family, the genera Roseburia and Blautia have been seen as being related to the maturation of the immune system or intestinal inflammation [97,98]; E. hallii, which interacts with other SCFA-producing bacteria, provides another example [99]. Roseburia intestinalis and acetogenic species work together in a cooperative manner to carry out butyric metabolism, which occurs without the production of H 2 [100]. On the other hand, when degrading L-fucose and fucosyllactoses, E. hallii engages in a trophic interaction with B. breve and B. infantis, highlighting the metabolic versatility of E. hallii, as it can utilize the intermediates produced during bifidobacterial oligosaccharide fermentation [101]. In the case of the Ruminococcaceae family, Faecalibacterium prausnitzii represents up to 5% of the fecal microbiota of healthy adults, being one of the most abundant butyrate-producing bacteria [102,103]. Different studies indicate the relationship between lower levels of this bacterium and various diseases, such as inflammatory bowel disorders (IBD) [104,105]. While most butyrate-producing microbes are classified under the Firmicutes phylum, research has indicated that some species from Actinobacteria, Fusobacteria or even Proteobacteria can also generate butyrate [106,107].
This finding adds to the evidence that, beyond carbohydrates, SCFAs can also derive from the fermentation of proteins and amino acids. Rasmussen et al. showed that the addition of albumin resulted in an increase in fecal C2-C5-SCFAs and that the incubation of fecal samples with specific amino acids resulted in the increase in specific SCFAs: hydroxyproline, serine, and glutamate resulted in an increased concentration of both acetate and butyrate, whereas histidine resulted in the increased production of acetate, and propionate was also produced from aspartate [108]. These findings were confirmed by Macfarlane et al., who found an increase in the concentration of SCFAs after the application of casein and bovine serum albumin to washed-gut bacteria, obtained from fresh faeces [109].
Although the main SCFAs are acetate, propionate and butyrate, other minor ones such as formate and lactate are also produced as a result of microbiota metabolism. In this way, the producing bacteria of these SCFAs and their role in the physiology of the host are drawing attention.
Formate appears as one of the end products of Bifidobacteria metabolism consuming HMOs, as occurs with acetate, lactate and 1,2-propanediol [77]. The formate can derive from an inadequate metabolism of LnNT, one of the main HMOs, by B. infantis, which leads to the production of formate and ethanol, instead of acetate and lactate [110]. E. hallii has also been identified as a format-producing bacteria. In this case, the production is carried out from 1,2propanediol, obtaining both formate and propionate and butyrate [101,111]. This example further shows the relationship between SCFA-producing bacteria and their metabolisms.
Besides, as previously indicated, lactate is one of the metabolites produced in the early stages of life, along with formate and acetate. Therefore, lactate-producing bacteria are related to the first stage of the intestinal microbiota, mainly Bifidobacteria and Lactobacilli, but also Staphylococci and Streptococci [112,113]. Lactate can be used by the bacteria present in gut microbiota, such as E. hallii and Anaerostipes caccae, which produce propionate and butyrate as a result [85,86]. Although the main SCFA-producing bacteria have been described, further research is needed in this field. The development of new methodologies and the use of novel approaches, mainly whole-genome sequencing and metabolomics, will allow for the identification of specific SCFAs-producing bacteria and also increase knowledge of the interactions between them and our organism, and the related consequences for human health.

SCFAs Production as a Marker of Healthy Gut Ecosystem
SCFAs production is essentially a biochemical process carried out by gut bacteria, but it may reflect the homeostasis of the gut. Several lines of evidence support the concept of SCFA production as a marker of a healthy gut ecosystem.
First, SCFAs-producing taxa are usually commensal, beneficial bacteria, with a direct positive effect on the gut barrier and immunity beyond the production of SCFAs [55,114].
Moreover, the mucus colonic layer is another element that is positively influenced by SCFAs.
The health of the mucus barrier can be considered a result of host-microbiota and within-taxa microbiome cross-talk, and these interactions also influence the production of SCFAs. As an example, the phenomenon of cross-feeding allows for taxa such as Akkermansia muciniphila, which is able to use the carbohydrates included in the mucus as a source of energy, to release oligosaccharides and acetate in the intestinal microenvironment and feed other bacteria [115,116]. Those molecules are then captured by bacteria such as Eubacterium hallii, which then can produce propionate, butyrate and vitamin B 12 , which are released in the lumen and can exert their trophic effects on colonocytes [111,117].
Conversely, SCFAs, mainly butyrate, may influence both the quality and the quantity of the mucus produced by the intestinal goblet cells. The induction of mucus production by SCFAs can be assessed by measuring the expression of MUC2, a gene coding for mucin 2. In mice, butyrate enemas may enhance MUC2 expression in the colon, indicating an increase in its production, and can reduce mucus' thickness [118]. Butyrate may enrich the mucus layer, improving the processes of sulphation, acetylation, and sialylation, and creating different kinds of mucins that can be used as substrates for different metabolic pathways by the gut bacteria [119]. Sialylation plays a key role in mucus homeostasis, and its defects are linked to inflammatory diseases [120]. Moreover, butyrate favors the adherence of Bifidobacteria to the epithelial barrier, reducing the adhesion of potentially pathogenic species such as E. coli [121]. This hypothesis is strengthened by the evidence of SCFA imbalances in disease conditions. As an example, in ulcerative colitis (UC), both detrimental shifts in the gut microbiome (and, more specifically, a reduction in SCFA-producing bacteria) and alterations in the mucus layer can be found [122]. Although the pathogenesis of UC and other chronic conditions is still poorly known, the complex relationship between the gut microbiome, SCFAs, colonocytes and intestinal mucus appears to be a promising therapeutic target, and deserves further research [104,123].

Mechanistic Involvement of SCFAs in the Development of Human Diseases
SCFAs play a significant anti-inflammatory role in the regulation of immune function [58], taking part in the prevention of various inflammatory chronic disorders [104,[124][125][126].
In gut diseases, both acute and chronic inflammation are relevant. Transient acute inflammation, an essential defense mechanism of the immune system against injurious stimuli, is of particular relevant [127,128]. In this condition, when cells are damaged, instead of directly targeting the injurious stimuli, such as any invading viruses or bacteria, Nutrients 2023, 15, 2211 7 of 22 the immune system will use the "self-destroy and rebuild" strategy, targeting the damaged cells. By using a programmed cell death such as pyroptosis [129] and necroptosis [130] to actively destroy the cells, stimuli such as viruses or bacteria are also effectively cleared. On the other hand, chronic inflammation develops when the stimulus cannot be removed and is associated with diseases like IBDs, where SCFAs play a key role [131].

SCFAs and IBD
IBD includes chronic inflammatory disorders of the gastrointestinal tract associated with a gut microbiota imbalance. Patients with IBD are known to share, compared with healthy subjects, a reduction in butyrate producers of the Firmicutes phylum, mainly Roseburia spp and Faecalibacterium prausnitzii, and an increase in opportunistic bacteria [104,132]. In addition to a reduced SCFAs production, the uptake and oxidation of butyrate appears to be inhibited in patients with UC [133]. This leads to a weakening of their anti-inflammatory activity, thus promoting disease progression. More specifically, propionate and butyrate stimulate T-reg proliferation and function through GPR-43 pathways and HDACs' inhibition [134][135][136]. SCFAs also lead to a downregulation of proinflammatory cytokines levels because of the inhibition of NF-κB and HDCAs activity [137][138][139], and to an increase in the anti-inflammatory ones through GPCRs [60].

SCFAs and Colorectal Cancer (CRC)
CRC is a multifactorial disease and the gut microbiota play an important role in its development [143]. Patients with CRC showed an increase in pathogenic bacteria (e.g., Fusobacterium nucleatum) and a depletion in butyrate producers [138,144,145]. The reduced production of SCFAs leads to a pro-inflammatory environment, which can contribute to the initiation and progression of CRC [146]. In addition, butyrate can change redox state and D-glucose metabolism, enhancing cancer cells' apoptosis [147], while the inhibition of HDCAs regulates the expression of p21, arresting cell cycle and consequent cancer proliferation [148]. Proliferation is also inhibited by propionate via GPR-43, which is often lost in colon cancer cells [124].

SCFAs and Cardiovascular Diseases (CVDs)
There is a large body of evidence suggesting that SCFAs play a role in the pathogenesis of CVDs, a group of disorders that include hypertension and atherosclerosis. A reduction in butyrate producers in the gut microbiota and the deficient intestinal absorption of SCFAs have been observed in patients with hypertension [149,150]. Moreover, SCFAs appear to have a dual effect on the regulation of blood pressure. For example, when binding Olfr-78, acetate and propionate lead to renin release, increasing blood pressure [151]. By contrast, when binding GPR-41, they reduce blood pressure via vasodilatation [152], which is also obtained by the effect of butyrate on afferent vagal terminals [153]. In atherosclerosis, a similar pathway has been noted [154], as SCFAs, mainly butyrate, appear to play a protective role in the regulation of inflammation and stabilization of plaques by downregulating the expression of CCL-2, VCAM-1, and MMP-2, resulting in the lower migration of macrophages, increased collagen deposition and ultimate plaque stability [155].

SCFAs and Metabolic Diseases
As anticipated above, SCFAs regulate metabolic pathways and food intake, thereby playing a role in the development of metabolic diseases. Obesity is associated with an imbalance in the gut microbiota, mainly an increased Firmicutes/Bacteroidetes ratio, and an increase in fecal-SCFAs [126,156], although circulating SCFAs are reduced [157]. Type 2 diabetes (T2D) is instead characterized by a decrease in butyrate producers in the gut microbiota [158]. Normally, SCFAs moderate food intake, stimulating the secretion of satiety hormones such as PYY and GLP-1 via GPR-41 and GPR-43 [159,160] and through the inhibition of HDACs [161]. Furthermore, acetate can cross the blood-brain barrier, causing a decreasee in appetite [45]. SCFAs can also improve glucose homeostasis in an AMPK-dependent manner involving PPARγ-regulated effects on gluconeogenesis and lipogenesis [48]. Moreover, propionate enhances glucose-stimulated insulin release via GPR-43 and increases β-cell mass [162]. SCFAs can stimulate adipocyte differentiation [163,164] and decrease lipid plasma levels through the inhibition of lipolysis and stimulation of lipogenesis [165][166][167] and cholesterol plasma levels, enhancing its hepatic uptake [168].
Overall, these mechanistic pathways of SCFAs in different disorders pave the way for the therapeutic use of SCFAs in clinical practice. Table 1.

Therapeutic use of SCFAs in Clinical Practice
Considering the involvement of SCFAs in the colon physiopathology, and also considering the effects those molecules have at the cardiovascular and metabolic levels, many studies have investigated the potential of SCFAs as a therapeutic option for both intestinal and cardiometabolic disorders.
Several lines of evidence for the use of SCFAs in intestinal disorders derive from mouse models. Butyrate enemas were effective in improving symptoms, inflammation and the sodium absorption of colonocytes in mice with experimental distal colitis [169]. However, these pre-clinical findings were not replicated in humans, regardless of the disease.
In a randomized placebo-controlled trial of 91 patients, where enemas of acetate, propionate and butyrate were used to treat left-sided UC, SCFAs were not more effective than placebo in improving the clinical picture, only levels of mucin depletion before and after treatment [170].
In a recent systematic review of randomized controlled trials using butyrate enemas in IBD, the study concluded that, for butyrate, enemas are not effective in UC, while for Crohn's disease, more studies are needed to clarify the role of SCFAs [171].
Oral butyrate supplementation was also valued for IBDs. In UC patients, sodium butyrate microcapsules were effective in reducing the Mayo score and faecal calprotectin levels compared to mesalamine alone [172]. Thus, in a bigger, randomized, placebocontrolled study conducted on paediatric patients, there was no significative difference between the standard-of-care therapy and the addition of sodium butyrate oral capsules in both newly diagnosed Crohn and UC [173].
In a systematic review of SCFAs enemas treatment for late-radiation proctitis, patients experienced a significant decrease in the weekly episodes of rectal bleeding and consequently higher levels of haemoglobin in only one study, without relevant results in other studies [174,175].
For IBS, data from in mouse studies show conflicting results about the role of SCFAs in the pathogenesis and physiopathology of this condition and its influence on visceral hypersensitivity [176][177][178]. Nevertheless, in human studies, a reduction in SCFA-producing bacteria was demonstrated for IBS-D and IBS-M [179], and the administration of butyrate in a triglyceride matrix was significantly effective in reducing the clinical severity of this disease [180]. However, double-blind, randomized, placebo-controlled studies are necessary to better evaluate the impact of this supplementation for IBS.
A prospective placebo-controlled randomized study demonstrated a significant impact of butyrate microcapsules in reducing episodes of diverticulitis in patients with diverticular disease [181]. These promising but preliminary findings support further research in this field.
Several pre-clinical lines of evidence that support the role of SCFAs in cardiometabolic disorders have recently been revealed. In mice fed a high-fat diet, the addition of butyrate was associated with a reduction in hepatic lipogenesis and hepatic steatosis, as well as with improvements in hepatic function and lipid profile, suggesting a possible role for SCFA supplementation in NAFLD [182].
In another mouse model, butyrate reduced heart ischemia-reperfusion damage [183], improved vascular function and reduced tension [184][185][186]. However, as in gastrointestinal disorders, these promising pre-clinical findings were not replicated in human studies [187]. Oral therapy with butyrate was not effective in improving metabolic outcomes in patients with diabetes [188] or with metabolic syndrome [189].
One reason for these unsatisfactory results is the therapeutic formulation of SCFAs, as butyrate supplements cannot reach high concentrations in the gut when administered by mouth. Recently, the colonic-delivery formulation of butyrate was found to positively affect quality of life and the gut microbiota composition of patients with UC [190], suggesting that this approach deserves further research.

How to Foster the Production of SCFAs in the Human Gastrointestinal Tract
Diet can influence and modulate the gut microbiota of individuals [191]. The major constituents of a normal diet, called macronutrients, are carbohydrates, protein, and fats. Fibers, defined as nondigestible carbohydrates composed of more than three monomeric units, could be considered the "fourth macronutrient"; fermentable fibers are used as substrates by colonic microbes, from which they are transformed into various metabolites including SCFAs [192]. Dietary fiber, prebiotic fiber supplements, and probiotics can modulate the gut microbiota and increase the overall production of SCFAs, as shown in Figure 2.

Diet
Even if there is not high homogeneity between studies on this issue, there is a general agreement that diets rich in fiber increase the amount of SCFA, in particular, acetate and butyrate [193]. Several studies reported that dietary interventions could increase the SCFA-producing bacteria. Studies focusing on the effects of high-fiber diets, such as Mediterranean, vegetarian, and vegan diets, on patients affected by IBD, resulted in improvements in microbiome outcomes (increase in alpha diversity, increase in specific microbial population, etc.), in laboratory exams (reduction in C-reactive protein (CRP), reduction in fecal calprotectin, etc.), and IBD-specific outcomes (i.e., reduction in Mayo Score), as well as in an increase in SCFA levels [194]. Likewise, a high-fiber diet led to a significant reduction in glycated hemoglobin, and an increase in glucagon-like peptide-1 (GLP-1) production, Bifidobacteria count, and total SCFA amount in patients affected by T2D [195]. On the other hand, studies evaluating the use of ketogenic diet (a lowcarbohydrate, high-fat diet able to induce physiological ketosis) showed a decrease in beneficial bacteria (i.e., Bifidobacteria, Eubacterium rectale, Roseburia) and total bacterial count and abundance [20]. As a consequence, a ketogenic diet may induce a reduction in both total SCFAs and their single components [196].

Prebiotics
Prebiotics, defined as «substrates that are selectively utilized by host microorganisms conferring a health benefit», are widely used to increase SCFA levels [197]. Some studies showed an increase in total SCFAs, butyrate, acetate, and propionate, using arabinoxylan oligosaccharides (AXOS) at high doses (above 7.5 g per day) [198], and similar results were obtained if AXOS was administered through fiber-enriched food [199,200]. Studies performed on healthy people using different prebiotics, such as xylooligosaccharides, inulin, resistant starch, raffinose, and galactooligosaccharides, did not show any impact regarding SCFA levels [201]. In patients with T2D, resistant starch seems to increase SCFA levels, leading to a reduction in intestinal permeability, inflammation, and circulating cytokines [202]. The increase in SCFAs was also observed in patients during enteral

Diet
Even if there is not high homogeneity between studies on this issue, there is a general agreement that diets rich in fiber increase the amount of SCFA, in particular, acetate and butyrate [193]. Several studies reported that dietary interventions could increase the SCFA-producing bacteria. Studies focusing on the effects of high-fiber diets, such as Mediterranean, vegetarian, and vegan diets, on patients affected by IBD, resulted in improvements in microbiome outcomes (increase in alpha diversity, increase in specific microbial population, etc.), in laboratory exams (reduction in C-reactive protein (CRP), reduction in fecal calprotectin, etc.), and IBD-specific outcomes (i.e., reduction in Mayo Score), as well as in an increase in SCFA levels [194]. Likewise, a high-fiber diet led to a significant reduction in glycated hemoglobin, and an increase in glucagon-like peptide-1 (GLP-1) production, Bifidobacteria count, and total SCFA amount in patients affected by T2D [195]. On the other hand, studies evaluating the use of ketogenic diet (a lowcarbohydrate, high-fat diet able to induce physiological ketosis) showed a decrease in beneficial bacteria (i.e., Bifidobacteria, Eubacterium rectale, Roseburia) and total bacterial count and abundance [20]. As a consequence, a ketogenic diet may induce a reduction in both total SCFAs and their single components [196].

Prebiotics
Prebiotics, defined as «substrates that are selectively utilized by host microorganisms conferring a health benefit», are widely used to increase SCFA levels [197]. Some studies showed an increase in total SCFAs, butyrate, acetate, and propionate, using arabinoxylan oligosaccharides (AXOS) at high doses (above 7.5 g per day) [198], and similar results were obtained if AXOS was administered through fiber-enriched food [199,200]. Studies performed on healthy people using different prebiotics, such as xylooligosaccharides, inulin, resistant starch, raffinose, and galactooligosaccharides, did not show any impact regarding SCFA levels [201]. In patients with T2D, resistant starch seems to increase SCFA levels, leading to a reduction in intestinal permeability, inflammation, and circulating cytokines [202].
The increase in SCFAs was also observed in patients during enteral nutrition treated with fiber-enriched formula, with a general reduction in diarrheal events [203,204].

Probiotics
Since the modulation of gut microbiota is a key way to induce a boost in SCFA levels, the administration of probiotics could be the most effective strategy. Both in vitro and in vivo studies confirmed a positive role of probiotics in increasing SCFAs [205]. The most used probiotics are those of Lactobacillus genera, mainly Lactobacillus plantarum [206,207], Lactobacillus paracasei [207], and Lactobacillus rhamnosus [208]; all the above studies confirmed, in animal models or in healthy volunteer humans, an increase in Bifidobacteria and in other beneficial microbes, leading to an increase in total SCFAs [206][207][208]. Other studies, using different probiotics species to enrich the beneficial bacteria count as well as the SCFA amount, obtained the same results in different disease settings, such as colorectal cancer, obesity, T2D, respiratory tract, and cardiovascular diseases [201,205,209,210].

Fecal Microbiota Transplantation
Fecal microbiota transplantation (FMT) is the transfer of feces from a healthy donor into the gut of a recipient to cure a disease associated with gut microbiome imbalance. FMT is a well-established therapy for recurrent Clostridioides difficile infection [211] and its complications [212], and has been investigated in other conditions with promising results [213,214].
Several recent lines of evidence suggest that that the increase in SCFAs may be a key determinant of FMT success in different diseases. First, in a mouse model of ischemic stroke, mice transplanted with feces rich in SCFAs, mainly butyrate, experienced an amelioration in neurological symptoms [215].
Moreover, in a randomized controlled trial where FMT from donors with balanced microbiome was more effective than placebo in reducing IBS-related symptoms, FMT increased the fecal SCFA levels, and the post-FMT increase in butyrate levels correlated inversely with symptoms [216]. Finally, in a small pilot trial, FMT from mixed lean donors was able to increase the levels of SCFAs-producing bacteria [217].
Overall, these findings suggest that FMT may be a therapeutic pathway to increase SCFAs in the recipient, especially with the use of targeted donors [218]. However, the real advantage of this therapeutic strategy in clinical practice has yet to be confirmed.

Conclusions
SCFAs, mainly butyrate, acetate and propionate, play several key roles in human health, from the modulation of the immune system to the regulation of metabolic pathways and the restoration of the gut barrier. Several bacteria are able to degrade substrate sources (mainly non-digestible polysaccharides, but also, less frequently, proteins) to produce SCFAs. The production of specific SCFAs changes over the lifetime, depending on the variations in our diet and consequent shifts in our gut microbiome. SCFAs have been investigated as a therapeutic option in several disorders, with promising findings in pre-clinical models but without satisfactory findings in humans due to the poor colonic availability of oral SCFAs. Moreover, the increase in SCFAs appears to be one of the key therapeutic pathways in several approaches aiming to modulate the gut microbiome, including diet, prebiotics, probiotics, or FMT. Further research, aiming to increase our knowledge of the role of SCFAs in human disorders, investigate new delivery formulations for SCFAs, and disentangle the value of SCFAs as a therapeutic pathway of microbiome modulators, are advocated.